Reference electrode assembly

ABSTRACT

A modular reference electrode assembly having a constrained-diffusion liquid junction between a liquid junction solution and a sample solution having separate flow paths, said assembly comprising a flow cell having attached thereto a constraint comprising a region of porous material permeable to water and salts; a remote reservoir for holding said liquid junction solution; a means for moving said liquid junction solution from said reservoir to said constraint; and a reference contact region is provided. This unique configuration provides a reference electrode device of the constrained-diffusion liquid junction type useful in pH and/or ion-selective electrode (ISE) potentiometric sensors and is particularly suitable for use in a mini-integrated electrochemical analyzer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/921,103, filed Aug. 29, 1997 which is a continuation-in-partof U.S. patent application Ser. No. 08/552,833, filed Nov. 3, 1995.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] N/A

FIELD OF THE INVENTION

[0003] This invention provides a reference electrode device of theconstrained-diffusion liquid junction type useful in pH and/orion-selective electrode (ISE) potentiometric sensors and is particularlysuitable for use in a mini-integrated electrochemical analyzer.

BACKGROUND OF THE INVENTION

[0004] Conventional types of reference electrodes have a liquid junctionwhere the sample meets the junction solution. The junction is typicallyeither open or constrained. In an open junction system, the liquidjunction operates by free diffusion. In a constrained-diffusion junctionsystem, a region of porous material permeable to water and salts (amembrane, porous plug, frit, or the like) is placed at the site of theliquid junction. The porous material acts as a constraint wherebypassage of large molecules (such as protein) and bulk liquid transportis generally hindered.

[0005] The liquid junction solution (also commonly referred to as thesalt bridge solution) typically contains a solution saturated with asalt (such as an equitransferent salt, including KCl, KNO₃) whichfunctions to reduce and maintain constant the interfacial potentialwhich develops across the liquid junction boundary, typically referredto as a liquid junction potential. The difference in liquid junctionpotentials between the system calibrator and the sample is referred toas the residual liquid junction potential. Typically, the residualliquid junction potential increases as the ionic strength differencebetween the system calibrator solution and the sample solutionincreases. The residual liquid junction potential is generallyconsidered to compromise the accuracy of the associated potentiometricsensors and therefore a multi-use reference electrode is typicallydesigned to minimize the residual liquid junction potential for as longas possible, while balancing other design constraints.

[0006] In potentiometric systems that are designed with miniaturizedworking or indicator electrodes (typically pH and/or ion selectiveelectrodes), the necessity of the junction solution makesminiaturization of a reference electrode difficult. Further, for thereference electrode to have a multiple use capability, the liquidjunction solution must be present in a volume and concentrationsufficient to minimize the residual liquid junction potential over itsuseful lifetime. This requisite volume particularly complicatesminiaturization of constrained diffusion liquid junctions since thevolume must typically be maintained close to the constraint to minimizeproblems associated with excessive ion depletion through the constraint.Another drawback of this type of reference electrode is that it tends tobe orientation dependent. During operation, an upright orientationgenerally must be maintained with the liquid junction solution on top ofthe constraint in order to maintain contact therebetween. Moreover, inorder to minimize errors due to the residual liquid junction potential,the junction solution is generally saturated with the equitransferentsalt.

[0007] Conventional reference electrodes utilize a reference contactregion (also sometimes referred to as electrode elements) immersed in astagnant portion of the junction solution which contains a constantconcentration of the equitransferent salt. The reference contact regionis often silver based, consisting of an electrochemically reversibleredox electrode couple such as Ag/Ag⁺ and Ag/AgCl. When salt solutionsare used with silver based reference contact regions such as Ag/AgCl,the AgCl is susceptible to dissolution. In constrained-diffusion typeliquid junctions, this dissolution is problematic because it leads tosubsequent precipitation of silver salts on the region of porousmaterial constraint, thus leading to undesirable fouling of theconstraint, which in turn generally results in an erratic referenceelectrode performance.

[0008] Commonly, because of the above-described fouling problemassociated with the use of silver based reference contact regions,barrier membranes have been used to restrict Ag⁺ ion migration from thereference contact region to the porous material constraint region. Theuse of a barrier membrane, however, carries with it inconveniencebecause the first use wet-up of the reference electrode is hindered bythe barrier membrane, thus requiring a long soaking time in the junctionsolution prior to the first use. In addition, any additional component,such as this barrier membrane, tends to complicate miniaturization ofthe reference electrode.

[0009] Another common problem associated with the use of saturatedequitransferent salt solutions in constrained-diffusion type of liquidjunction reference electrodes is that when the reference electrode isstored and/or used at sub-ambient temperatures, salt crystallization andprecipitation may occur between the reference contact region and theregion of the porous material at the junction, which in turn leads toerratic reference electrode potentials. An additional problem associatedwith the use of a saturated equitransferent salt solution is that thesaturated solution may contribute to reproducibility and/or accuracyproblems with blood samples because of interference caused byprecipitation of protein and crenation of red blood cells present in thesample.

[0010] Some of the aforementioned drawbacks associated with constraineddiffusion liquid junctions are reduced or absent in open (freediffusion) liquid junctions. In this regard, reservoirs for holding ofjunction solutions have been described for open junction type referenceelectrodes, where there is no region of porous material to act as aconstraint at the junction. For example, A. K. Covington et al. (Anal.Chim. Acta, 1985, 169, pp. 221-229) describe a open junction where thejunction solution is moved from a KCl reservoir via a syringe. In thisprior art system, the liquid junction is established with each sample,but because this type of system leads to cross-contamination of theliquids upon use, the junction solution must be discarded along with thesample thus leading to increased waste. Moreover, such contaminationtends to dictate placement of the reference electrode downstream in thesample path of all working electrodes. This restrictiondisadvantageously limits flexibility of analyzer configuration.

[0011] Moreover, open free-diffusion liquid junction referenceelectrodes tend to require specific orientations and geometries toprovide good reproducible junctions. Reproducibility for openfree-diffusion liquid junctions tends to depend on uniform, generallycircular junction geometries and a constant orientation. (See, e.g., R.E. Dohner et al. Anal. Chem., 1986, 58, pp. 2585-2589; T. R. Harbinsonet al., Anal. Chem., 1987, 59, pp. 2450-2456). The orientation andgeometries requirements are particularly limiting when attempting toadapt such a reference electrode to a miniaturized, modular system inwhich it may be desirable to locate the reference electrode within anarray of working electrodes and utilize componentry and dimensionscommon to those working electrodes. The aforementioned orientationrequirement may be particularly troublesome in the event a miniatureportable analyzer is desired for use in locations where the electrodearray may be utilized in various or unstable orientations, such as maybe encountered in the field or in mobile applications such as on boardambulances, ships and/or aircraft.

[0012] Thus, although many different reference electrodes are known inthe prior art, there is a need to discover alternative referenceelectrodes for electrochemical analyzers, particularly modular referenceelectrodes that may be easily adapted for use in a mini-integrated typeof analyzer.

SUMMARY OF THE INVENTION

[0013] Many of the shortcomings associated with prior art referenceelectrodes have been overcome with the discovery of a referenceelectrode assembly of the present invention.

[0014] According to the invention, provided is a modular referenceelectrode assembly adapted for serial integration within an orientationindependent array of working electrodes, the working electrodes disposedin a plurality of electrode receiving positions on a support member ofan integrated sample analyzer. The reference electrode includes:

[0015] a flow cell of modular construction sized and shaped forselective disposition in any of the plurality of electrode receivingpositions on the support member, wherein said flow cell is adapted forbeing serially retained within a sample flow path of the array;

[0016] a flow cell of modular construction sized and shaped to interfitbetween guide members adapted to retain the array of miniaturizedworking electrodes, wherein the flow cell is adapted for being seriallyretained within a sample flow path of the array;

[0017] a liquid junction disposed within the flow cell;

[0018] a remote reservoir for holding a liquid junction solution, theremote reservoir being connected to the flow cell by a liquid junctionflow path, the liquid junction flow path being separate from the sampleflow path;

[0019] a reference contact region in physical contact with the liquidjunction solution;

[0020] the liquid junction including a constraint having a region ofporous material permeable to water and salts, the constraint having acontact portion adapted for contacting the liquid junction solution onone side thereof and a sample solution on an other side thereof;

[0021] the constraint adapted to substantially prevent bulk flow of theliquid junction solution therethrough, and to provide an orientationindependent, stable liquid junction; and

[0022] means for moving the liquid junction solution from the reservoirto the constraint and for applying pressure sufficient to maintain theliquid junction solution in contact with the constraint at substantiallyany orientation of the array of electrodes wherein the flow cell isoperable at substantially any orientation.

[0023] Also provided is a method for providing a modular, orientationindependent reference electrode for a system for potentiometricquantitative analysis. This method includes:

[0024] providing a flow cell of modular construction sized and shapedfor selective disposition in any of a plurality of electrode receivingpositions on a sensor support member adapted to support an array ofworking electrodes, wherein the flow cell is adapted for being seriallyretained within a sample flow path of the array;

[0025] interfitting the flow cell in series with the array ofminiaturized working electrodes;

[0026] moving a sample solution through the sample flow path over atleast one miniaturized working electrode disposed within the array;

[0027] moving the sample solution to the flow cell to form a liquidjunction between the sample solution and a liquid junction solution overa porous constraint wherein the sample solution is disposed in contactwith one side of the porous constraint and the liquid junction solutionis disposed in contact with an other side of the porous constraint, theconstraint being adapted to substantially prevent bulk flow of theliquid junction solution therethrough and to provide an orientationindependent, stable liquid junction, the liquid junction solutioncomprising a non-saturated equitransferent salt and being stored in aremote reservoir wherein the liquid junction solution is pumped to theconstraint per sample cycle and the liquid junction solution iselectrically connected with sensing equipment by a reference contactregion in physical contact with the liquid junction solution;

[0028] maintaining the liquid junction solution in contact with theconstraint with sufficient pressure wherein the flow cell is operable atsubstantially any orientation of the array of electrodes; and

[0029] measuring an electric potential developed between the referencecontact region and the working electrode.

[0030] The inventive reference electrode assembly is particularly usefulbecause it is not orientation and gravity specific. The modularconstruction enables it to be interchangeably retained at substantiallyany predetermined location within an array of working electrodes in aconfiguration similar to that utilized for the working electrodes.Moreover, the present invention may be fabricated as a multiple usereference electrode for use in a mini-integrated electrochemicalanalyzer having planar miniaturized working electrodes becausesufficient liquid junction solution is present for multiple uses withoutpresenting a space problem since the extra liquid junction solution isstored in the reservoir that is remote from where theconstrained-diffusion liquid junction is created. Other advantages ofpreferred embodiments are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] As described in more detail in the Examples section herein, FIG.1 is a schematic view of an embodiment of a reference electrode assemblyof the subject invention; and

[0032]FIG. 2 is a plan view of a portion of an array of workingelectrodes, including a flow cell of the reference electrode of FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] According to the invention, reference electrode 5 has a flow cellor front end 4 where the liquid junction solution meets the samplesolution at a junction constrained by a region of porous material orconstraint 18 permeable to water and salts. The porous materialgenerally hinders passage of large molecules (such as protein) and bulkliquid transport. Such porous materials have been used extensively inthe art as the “constraint” in constrained-diffusion liquid junctionreference electrodes and thus are easily recognizable by those skilledin the art and are widely commercially available. Examples of suchmaterials are porous plugs, frits and/or membranes. When selected as theconstraint material, porous membranes may be fashioned from suchmaterials as cellophane, cellulose acetate, partially nitrated cellulosepolycarbonate, combinations thereof, and so on. Particularly preferred,because of ease of use, cost and availability, is a cellophane membrane.

[0034] Flow cell 4 may be fashioned from any suitable material conduciveto the overall design of analyzer 1 such that the flow cell material iscapable of attachment to the constraint material, as well recognized bythose skilled in the art. If a reference contact region 13 is configuredas a part of the flow cell, then the material selected should benon-conductive. If the reference contact region of the referenceelectrode assembly is not a portion of the flow cell, then the materialselected may be conductive, if desired.

[0035] Attaching the constraint region of porous material to the flowcell where the junction is formed may be accomplished by any number ofmethods within the skill of one acquainted with the art, including forexample, bonding or attaching with welding, adhesives, mechanicalcompression and the like. The attachment should be secure enough tosubstantially prevent regions of dead volume in the junction solution.Preferably a hermetic seal is formed between flow cell 4 and constraint18. The area of the porous material exposed to the sample and/or thejunction solution may vary, spanning from the entire flow cell to only asmall portion of the flow cell, depending upon the specifications of theanalyzer design, and so on. Flow cell 4 can optionally be designed suchthat the porous material covers a distinct chamber or chamber region 19of the flow cell, where the constraint covers the chamber region and theliquid junction is formed over the chamber region.

[0036] A remote reservoir 10 of the invention functions as a storageunit for excess junction solution. According to the invention, thereservoir is external to flow cell 4 and, thus, external to theconstrained-diffusion region where the liquid junction is formed. Theexternal nature of the reservoir provides sufficient volume of liquidjunction solution for a multiple use reference electrode withoutrequiring a bulky storage unit for the junction solution at theconstrained liquid junction itself to thereby facilitate provision of aminiaturized, modular construction, including an orientationindependent, stable liquid junction as will be discussed in greaterdetail hereinafter. Throughout this disclosure, the term “stable liquidjunction” shall be defined as a liquid junction capable of generatingoutputs that are constant from sample to sample within about 0.14 mV(millivolts).

[0037] Surprisingly, however, this location of the bulk of the volume ofliquid junction solution remotely from constraint 18 did not generatethe ion depletion problems commonly expected with conventionalconstrained diffusion junctions. This is particularly surprising inlight of the reduced concentration of liquid junction solution utilizedto facilitate sub-ambient storage, as will be discussed hereinafter. Thereservoir may be designed in various sizes and shapes, depending uponthe design of the overall analyzer system and the desired lifetime ofthe reference electrode assembly. For example, the reservoir could beformed by holding the junction solution in the typical tubing (or tubingwith a bladder) leading to constraint 18 where the liquid junction isformed. Alternatively, the reservoir may be recognized as a separatecontainer connected by tubing or other such means to theconstrained-diffusion liquid junction. Appropriate separate structuresthat may be used as the reservoir include bottles or tanks and so on.

[0038] The liquid junction solution comprises an aqueous solution of asalt having equivalent cationic and anionic conductances, as are wellknown in the art and include equitransferent salts, such as KCl, KNO₃and equivalents thereof. Non-equitransferent salts, such as NaCl, NaBr,KBr, NaNO₃ equivalents thereof and so on may also be used, however,these lead to increased liquid junction potentials. Preferred areequitransferent salts (KCl, KNO₃, and mixtures thereof). Because of costand availability KCl is the most preferred salt. Other items mayoptionally be present in the liquid junction solution, including, forexample, compatible surfactants, or ions.

[0039] The use of silver ions in the liquid junction solution is usefulin the embodiments where the reference contact region is a silver basedmaterial. Preferably, the electrical contact region does not have abarrier membrane and the salt solution (preferably KCl) isnon-saturated. In this situation, the present invention utilizes ajunction solution further comprising Ag⁺ ions, the ions preferablypresent in an amount sufficient to establish stable potentials at thesilver based reference contact region but low enough so as to not inducefouling at the porous material constraint. It is known from the priorart that the concentration of Ag⁺ ions present at saturation isdependent on the concentration of the electrolyte salt present in thejunction solution (e.g., see Forbes, G. S., Journal of the AmericanChemical Society, 33, pp. 1937-46 (1911), hereby incorporated byreference). Preferably, the Ag⁺ ions are included in the junctionsolution to the point of saturation when the KCl solution is used at aconcentration of less than about 2 M. Preferably when the KCl is presentin a concentration of greater than about 2 M to saturation (4.2 M atroom temperature or 7.1 M at 100° C.), then the Ag⁺ ions are provided ina concentration below saturation from about 0.01 mM to about 1 mM, mostpreferably 0.6 mM. The Ag⁺ ions may be provided to the salt solution orformed in conjunction with the preparation of the junction solution byany number of familiar methods. For example, the junction solution maybe prepared from a dilution of a saturated KCl/saturated AgCl solution;addition of solid KCl to a 2 M KCl/saturated AgCl solution; dissolutionof appropriate quantities of KCl and AgNO₃ in water, and so on.

[0040] Any desired concentration of the equitransferent salt in thejunction solution may be utilized. Although saturated solutions of thesalt may be used, one of the advantages of the inventive assembly isthat the salt may be used in a concentration below saturation. In apreferred embodiment, a non-saturated solution of the salt is employed.Preferably KCl is selected as the salt and used in an amount rangingfrom about 0.5 M to about 7.1 M, more preferably 1 M to 4.5 M, and mostpreferably about 3.2 M. One of the advantages in using a non-saturatedsolution is particularly evident when a Ag/AgCl material is used as thereference contact region because with the non-saturated KCl solution,the Ag⁺ concentration present at saturation is lowered (thus assistingin minimizing AgCl dissolution). Another advantage in using anon-saturated solution is to facilitate sub-ambient usage and/or storagesince this eliminates the likelihood of KCl precipitation in the liquidjunction flowpaths.

[0041] The means for moving the liquid junction solution from thereservoir to the constraint region may be selected from various knowndevices, taking into effect the overall design of the analyzer. Includedin the definition of devices that may be selected for the means foractively moving the liquid are devices such as pumps, syringes, and thesort. For automated systems, a peristaltic pump 15, reciprocatingsyringe or other such device is particularly useful.

[0042] The design of the analyzer may be such that the device used formovement of the junction solution may also be used for moving otherliquid(s) in the analyzer, such as, for example, the sample solution,calibration solution, wash solution, combinations thereof, and so on.Alternatively, a device specific to movement of the liquid junctionsolution alone may be used. The flow rate and volume of the liquidjunction solution may be adjusted to the specifications of the analyzerto provide a concentration of electrolyte salt at the liquid junctionthat resembles that of the salt solution of the reservoir, asrecognizable to those skilled in the art.

[0043] Reference contact region 13 may be placed anywhere withinanalyzer 1 as long as it may function so that electrical contact is madebetween the sensing equipment and the liquid junction solution. Suitablematerials for the reference contact region are materials includingconductive metals, alloys thereof, and composites thereof havingacceptable conductive properties, as are easily recognized by thoseskilled in the art (i.e. materials that are capable of providing anelectrochemically reversible redox electrode couple). Particularlysuitable are silver based materials and calomel (Hg/Hg₂Cl₂). Morepreferably, silver based materials are used (e.g., Ag/Ag⁺ and Ag/AgCl).Although barrier membranes covering all or a portion of the electricalcontact regions may be used, for ease of use and to facilitateminiaturization, preferably no such barrier membrane is present inreference electrode 5. Ability of the present invention to operatesuccessfully without such a barrier membrane, without excessive foulingof constraint 18 was surprising in light of the teachings of the priorart. In this regard, various configurations of reference electrode 5were tested and shown to operate successfully without substantialdegradation of constraint performance over a uselife of at least 28days.

[0044] For ease of use, reference contact region 13 may be designed intomultiple conductive regions that are connected together to facilitateelectrical contact between the sensing equipment and the liquidjunction. For example, two regions may be used. In this design, a firstregion 11 is where the contact is made with the liquid junctionsolution, with the first region prepared from materials that are capableof providing an electrochemically reversible redox electrode couple.Second region 25 is directly connected to the first region but does notnecessarily touch the liquid junction and may be prepared using anysuitable electrically conductive materials (i.e. extending beyond thosematerials capable of providing an electrochemically reversible redoxelectrode couple). In this design, the function of the second region isto facilitate making electrical contact to the sensing equipment.Illustrative of this set up would be a first region 11 comprising aconductive wire connected to the liquid junction solution and a secondregion 25 that is a “pad” embedded or screen printed onto thenon-conductive flow cell 4 as shown. In this embodiment the wire wouldbe set into the pad in a perpendicular fashion. The electricalcontact(s) from the sensing equipment would touch the second region pad,thus providing indirect contact between the sensing equipment and theliquid junction solution.

[0045] The reference electrode assembly may be used in conjunction withone or more potentiometric indicator or working electrode(s) 6 whoseresponse depends upon analyte concentration. Ion-selective electrodesbased on solvent polymeric membranes are examples of such workingelectrodes. Examples of analytes that may be measured include, forexample, chloride, potassium, calcium, sodium, pH, bicarbonate,magnesium, and so on. Reference electrode 5 and a working electrode 6constitute individual galvanic half-cells which together comprise anelectrochemical cell which allows for potentiometric quantitativeanalysis. The reference electrode assembly is capable of providing asteady and stable potential sufficient for potentiometric analysis overthe clinically relevant range of ionic strengths, proteinconcentrations, and hematacrit levels. Optionally, the referenceelectrode assembly may be packaged together with mini-integrated workingelectrodes 6 stationed on a sensor module 9 such that the entireconfiguration is a single disposable unit.

[0046] It is to be understood that various modifications to theinvention will be apparent to and can readily be made by those skilledin the art, given the disclosure herein, without departing from thescope and materials of this invention. It is noted that the followingexamples given herein are intended to illustrate and not to limit theinvention thereto.

EXAMPLES

[0047] Many configurations of reference electrode 5 may be designed.FIGS. 1-2 illustrate a configuration of a reference electrode assemblyincorporated into a mini-integrated electrochemical analyzer 1 where thereference electrode has a lifetime of at least about one month withtypical clinical usage.

[0048] More particularly, in FIG. 1 the schematic view illustrates aflow cell 4 of a reference electrode 5 positioned serially within anarray 7 of a plurality of working electrodes 6. The electrodes are eachdisposed between a pair of guide rails 8 of a sensor module 9. Thejunction solution is pumped from a remote external reservoir 10 locatedapart from the liquid junction region of the reference electrode 5. Theorientation of the reservoir 10 is not gravity specific, and thus thereservoir may be placed anywhere that is convenient to the other itemsof the analyzer. From the sample entry 12 portion of the sensor array,the analyte containing sample is pumped along sample solution flow path20 through the sample chambers of various working electrodes 6 andthrough sample chamber 19 of flow cell 4 to a sample exit 14. Samplesolution flow path 20 and liquid junction solution flow path 22 areseparate paths. A common pump 15 is used for sample solution movementand junction solution movement. The junction solution is moved alongliquid junction solution flow path 22 from the remote reservoir 10 toflow cell 4 of the reference electrode 5 each time a sample is run. Themovement of the junction solution may be continuous but more preferablyoccurs only at such time as when a sample (or reference liquid such as acalibrator or quality control material) is present. Once used, thejunction solution may be discarded as waste or flow path 22 may beconfigured as shown to enable the junction solution to be recycled andpumped back to reservoir 10. Preferably the solution is recycled, thusextending the lifetime of the reference electrode and also eliminatingwaste. In a particularly preferred configuration, the liquid junctionsolution is re-circulated and pumped with the introduction of eachanalyte sample and becomes stagnant after the testing of the sample iscompleted. Preferably, only a small volume of the reference electrolytesolution is moved to flow cell 4 of the reference electrode 5 andoptionally recycled back to the external reservoir 10 per cycle, inconjunction with a small front-end or flow cell reference electrodeassembly particularly useful in a mini-integrated analyzer.

[0049] As shown in FIGS. 1 and 2, the liquid junction solution may bepumped from the reservoir 10 to an inlet tube 16 over the region ofporous material forming constraint 18. The porous material (preferably apermeable membrane) is attached to flow cell 4 by fastening a gasketover the material whereby a hermetic seal between the porous materialand the flow cell is formed. The area constrained by the porous materialis where the sample and the junction solution interface in the flow cellthus forming the liquid junction. The flow cell has a roundedelliptical, or preferably circular shaped sample chamber 19, theseshapes of the chamber being helpful in providing an effective washout ofthe entire chamber and also to minimize bubble trapping. Junctionsolution depletion occurs at a rapid rate once the junction solution isheld stagnant in the flow cell. Design of the surface areas and volumeof chamber 19 relative to the sample volume and hold time may bemanipulated to provide the desired performance. Preferably when such anelliptical shaped chamber is used, the constraint region is attachedthereto. Advantageously, unlike many open junctions discussedhereinabove which are typically geometry dependent, the liquid junctionformed by constraint 18 of the present invention is adapted for use withsuch elliptical or other non-circular shaped chambers. In a preferredembodiment, chamber 19 has a volume of from about 0.5 to about 17 μL,most preferably, a circular chamber has a volume of about 0.5 μL. Asdiscussed hereinabove, electrical contact region 13 has two parts. Thefirst region 11 is a Ag⁺ or Ag/AgCl wire that is imbedded in a secondcontact region 25 that is attached to the non-conductive flow cell. Thesecond region 25 has an exposed portion in the flow cell through whichthe electrical contact is made with the sensing equipment. The sample isremoved from the sensor array through the sample exit 14 as waste whilethe junction solution is recycled back to the reservoir 10 via the exittube 17.

[0050] In the embodiment shown in FIGS. 1 and 2, the reference electrodemost preferably employs a non-saturated KCl liquid junction solutionstored in a reservoir having a volume of from about 1 mL to about 50 mL(most preferably 8 mL), a membrane porous constraint region, and asilver based reference contact region without a barrier type ofmembrane. Advantages of this embodiment include a multiple-usecapability, with minimum fouling of the porous material region over theuse lifetime. The assembly may be stored as a dry reference electrodeassembly. The KCl junction solution may be released form the reservoirwhich then easily wets the flow cell upon first use, where bubbleformation at the membrane region (which typically accompanies a firstuse) is reduced. Also, in this preferred embodiment, the junctionsolution is re-circulated and thereby greatly minimizes the reservoirvolume necessary for multiple use and also reduces the amount of wastegenerated as compared to systems with one-use only solutions. Thisreference electrode also may be stored at sub-ambient temperatures witha reduced possibility of salt crystallization while providingreproducible results having an acceptable accuracy with multiple testingcapability. In this embodiment there is exhibited an adequate ionicstrength independence at relatively low junction solution concentration,as low as about 0.5 M KCl. This is unexpected based on the Hendersonequation for the liquid junction potential of two freely diffusingliquids.

[0051] As best shown in FIG. 2, flow cell 4 is modular to facilitatedisposition serially with electrodes 6 at substantially any convenientelectrode receiving location 23 along flow path 20 of sensor module 9.Once a convenient location is determined, analyzer 1, including theelectronic portion thereof, may be configured accordingly in a mannerfamiliar to those skilled in the art. In a preferred embodiment as show,flow cell 4 has nominally the same, relatively miniaturized footprint ordimensions as each working electrode 6 and thus may be interchangeablyplaced in any of the electrode receiving locations 23. As used herein,the term “footprint” shall be defined as a maximum dimension in at leasttwo orthogonal dimensions. As shown, flow cell 4 fits within guide rails8 in nominally the same manner and occupies the same dimension 21 in thedirection of flow path 20 as each electrode 6. In a preferredembodiment, this sample flow path dimension 21 is approximately 0.5 cmcenter to center as shown. In addition to permitting such locationalflexibility, this miniaturization advantageously enables determinationof analyte content using a relatively small sample. For example, thepresent invention enables a sample as small as 53 μL to completely fillsample path 20 within a complement of 13 working electrodes 6 plus flowcell 4 for analyte determination.

[0052] This uniformity or modularity also advantageously facilitates useof standardized componentry in analyzer 1, such as, for example, use ofa single pair of guide rails 8 in sensor module 9 to secure both workingelectrodes 6 and flow cell 4. An important additional aspect of thismodularity, as provided in part by use of remote reservoir 10, theconfiguration of flow cell 4 and use of constraint 18, is the ability offlow cell 4 to be physically located either upstream or downstream alongsample flow path 20, of one or more working electrodes 6. Thus, workingelectrodes 6 may be disposed within sensor module 9 on opposite sides offlow cell 4 as shown. This aspect advantageously provides flexibility ofdesign and use of analyzer 1. This flexibility may be useful in enablinga single analyzer design to accommodate various numbers of workingelectrodes. For example, guide rails 8 may be shortened or provided withblanks on either side of flow cell 4 in the event relatively few workingelectrodes 6 are desired, or alternatively, may be lengthened toaccommodate additional electrodes to analyze additional analytes.

[0053] Since both flow cell 4 and working electrodes 6 are orientationindependent, the entire array 7 may be operated at substantially anyorientation. This aspect advantageously facilitates use of the presentinvention in numerous mobile applications where it may be inconvenientto maintain the array in a level orientation. This feature also servesto provide additional flexibility in analyzer design by, for example,enabling constraint 18 and accordingly, inlet tube 16 and exit tube 17to be disposed either above, below or alongside sample flow path 20. Astill further advantage of the configuration of the present invention isthat the miniaturized modular design of sensor module 9 including flowcell 4 may be sufficiently simple and inexpensive to produce that it maybe conveniently disposed and/or recycled at the end of its useful lifeand replaced with a new or refurbished module.

Example 1

[0054] Reference flow cells were either machined from rigid acrylic orinjection molded using ABS plastic. For each flow cell constructed, theconstraint material was an ion and water permeable free-standing 0.001″thick cellophane (uncoated regenerated cellulose) film membrane obtainedfrom Flexel, Inc., Atlanta, Ga. The film was placed over an ellipticalshaped chamber 19 of the flow cell (the chamber having a volume ofapproximately 3.5 μL). The film was hermetically sealed to the chamberregion of the flow cell with compression from a gasket on top of theflow injection sensor module assembly. Sample solution and liquidjunction solution flowed past each other on opposites sides of thesurface of the cellophane film membrane at a flow rates of from about 5to about 100 μL/sec. A common peristaltic pump was used tosimultaneously move both solutions. Electrical contact was establishedvia an electrochemically plated 0.012″ Ag/AgCl or Ag wire located eitherwithin the remote reservoir solution or in the base of the flow cellimbedded in screen printed epoxy silver in a perpendicular fashion toextend into the inlet tube of the flow cell. The sensing equipment usedwas a HP 3457A digital multimeter. The total volume of the solution inthe junction reservoir ranged from approx. 8 mL to approx. 15 mL. Thejunction solution was moved from the remote reservoir to the flow celland past one surface of the cellophane membrane with every samplesolution. The junction solution was then either re-circulated back tothe reservoir or sent to waste after the measurement cycle. Electrodemeasurements were taken when both the sample and liquid junctionsolutions were stationary.

[0055] Various junction solutions were tested. The aqueous basedsolutions tested were (a) 1.5 M of KCl saturated with AgCl (approx. 0.25mM); (b) 2.0 M of KCl saturated with AgCl (approx. 0.6 mM); (c) 3.5 M ofKCl with 0.6 mM of AgCl; and (d) 4.0 M of KCl with 0.6 mM of KCl, wheresome of the solutions further comprised about 0.05 g/L of BRIG® 700(manufactured by ICI Surfactants, Wilmington, Del.). The wet up of afully dry system was established to commercially desirablespecifications in less than five minutes. Ionic strength dependence overa clinically significant range of 0.120 mM to 0.200 mM was evaluated.Reference potential changes of less than 0.5 mV were measured for allsolutions tested over a time period averaging approx. 1 month. Thisreference electrode was used together with separate planar ion-selectiveelectrodes sensitive for pH, potassium, ionized calcium, and sodium forthe analysis of these analytes.

Example 2

[0056] Reference electrodes were prepared as described in Example 1 withthe following exceptions.

[0057] The placement of the reference contact region (a Ag wire) wasvaried. A three dimensional sodium ISE working electrode (200 Seriesobtained from Ciba Corning Diagnostics Corp., Medfield, Mass.) wastested with three reference electrodes using a junction solution of 2MKCl/saturated with AgCl. The reference electrode used as the control wasa 200 Series Corning Diagnostics Corp. three-dimensional type ofreference electrode. The calibration reagents were that used on the CibaCorning Diagnostics Corp. 644 Instrument.

[0058] In Set A, the reference electrodes tested had a Ag wire locatedin the base inside the chamber. A reference electrode prepared with a Agwire in the base of the flow cell was tested over a period of 35minutes. The average within sample drift was −0.047 mV/sec. A referenceelectrode with a Ag wire in the remote reservoir was tested under thesame conditions and was found to have an average within sample drift of−0.001 mV/sec. The control 200 Series reference electrode had an averagewithin sample drift of −0.0004 mV/sec.

[0059] In Set B, the reference electrodes tested had a Ag wire locatedin a base outside the chamber. A reference electrode prepared with a Agwire in the base outside the chamber of the flow cell was tested over aperiod of 35 minutes. The average within sample drift was 0.001 mV/sec.A reference electrode with a Ag wire in the remote reservoir was testedunder the same conditions and was found to have an average within sampledrift of 0.0004 mV/sec. The control 200 Series reference electrode hadan average within sample drift of 0.001 mV/sec.

Example 3

[0060] Reference electrodes were prepared as described in Example 1 withthe exception that the chamber had a larger volume (16.8 μl) as comparedwith the chamber of Example 1 (which had a volume of 3.5 μl). A threedimensional sodium ISE working electrode (as described in Ex. 2) wastested with three reference electrodes using a junction solution of 2MKCl/saturated with AgCl. The reference electrode used as the control wasa 200 Series Corning Diagnostics Corp. three-dimensional type ofreference electrode. The calibration reagents were that used on the CibaCorning Diagnostics Corp. 644 Instrument. The reference electrodestested had a Ag wire located in a base inside the chamber. Theelectrodes were tested over a period of 35 minutes. The average withinthe sample drift was −0.012 mV/sec. When repeated the average within thesample drift was −0.028 mV/sec. The control 200 Series referenceelectrode had an average within sample drift of 0.001 mV/sec.

That which is claimed is:
 1. A modular reference electrode assemblyadapted for serial integration within an orientation independent arrayof working electrodes, the working electrodes disposed in a plurality ofelectrode receiving positions on a support member of an integratedsample analyzer, said reference electrode comprising: a flow cell ofmodular construction sized and shaped for selective disposition in anyof the plurality of electrode receiving positions on the support member,wherein said flow cell is adapted for being serially retained within asample flow path of the array; a liquid junction disposed within saidflow cell; a remote reservoir for holding a liquid junction solution,said remote reservoir being connected to said flow cell by a liquidjunction flow path, said liquid junction flow path being separate fromthe sample flow path; a reference contact region in physical contactwith said liquid junction solution; said liquid junction including aconstraint having a region of porous material permeable to water andsalts, said constraint having a contact portion adapted for contactingsaid liquid junction solution on one side thereof and a sample solutionon an other side thereof; said constraint adapted to substantiallyprevent bulk flow of said liquid junction solution therethrough, and toprovide an orientation independent, stable liquid junction; means formoving said liquid junction solution from said reservoir to saidconstraint and for applying pressure sufficient to maintain said liquidjunction solution in contact with said constraint at substantially anyorientation of said array of electrodes wherein said flow cell isoperable at substantially any orientation.